Structure and Function of Chromosomes and Related Protein-DNA Complexes

We wish to determine the various three-dimensional structures adopted by chromosomes at differing points in the cell cycle; to work out the physico-chemical basis of their stability; and to elucidate the relationship between chromosome structure and the functions of chromosomes in key cellular processes including cell division, recombination, gene expression, and replication.

We are currently carrying out studies in four specific areas: (a) Studies on the mechanism by which regulatory proteins are provided access to their target sequences in chromatin; (b) Studies on the mechanism by which RNA and DNA polymerases are able to elongate through their natural chromatin substrates; (c) Studies on the mechanism of histone-DNA recognition and analysis of nucleosome positioning signals in eukaryotic genomes; and (d) Three-dimensional reconstructions of mitotic chromosomes in yeast.

Research in each of these areas involves biochemical work in order to prepare samples to be analyzed, followed by physical and structural analysis. Samples are prepared using sophisticated HPLC methodologies, with material obtained from natural sources or reconstituted from purified components which may be synthesized chemically or enzymatically or may be produced by molecular cloning. Our biophysical and structural studies include methodologies such as steady-state and time-dependent fluorescence spectroscopy (lifetimes, polarization, energy transfer), dynamic light scattering, ultracentrifugation, electron microscopic imaging and tomography, and x-ray scattering and diffraction, together with novel enzyme-coupled assays for macromolecular dynamics.